In this study, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) rats. Using 2-D DIGE, we have obtained 2-D gel maps of 301 identified non-redundant proteins from rat aorta and observed 18 proteins that significantly change abundance with aging. Utilizing iTRAQ, 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Notably, proteomic analysis shows that one protein of interest, MFG-E8, significantly increases in abundance in old rat aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMC) of the thickened aged aortic wall. Exposure of early passage VMSC from young aorta to Ang II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMC from old rats. Treatment of VSMC with MFG-E8 increases MCP-1 and VSMC invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMC invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the Ang II MCP-1/VSMC invasion signaling cascade. Importantly, we have identified that aging arterial MFG-E8-enriched VSMC are activated and proliferating both in vivo and in vitro. Increased MFG-E8 in VSMC triggers phosphorylation of ERK1/2, augments levels of PCNA and CDK4, increases BrdU incorporation and promotes growth. The knockdown of MFG-E8 reduces rate of cell cycling, accelerating signaling molecules PCNA and CDK4 expression, facilitating cell entry into a growth-arrested state. Furthermore, we find that av5 and PDGF are upregulated with MFG-E8 and also are elements to relay proliferative signals to aging VSMC. In addition, exposure of rhMFG-E8 to early primary passage old VSMCs increases activated TGF-1 in a dose-dependent manner. Interestingly, rhMFG-E8 treatment also enhances downstream signaling molecular expressions of p-SMAD2/collagen I in old VSMCs. In contrast, the silencing of MFG-E8 substantially decreases TGF-1 activity, p-SMAD2, and collagen expression in both young and old VSMCs. Furthermore, TGF-1, SMAD2, extracellular matrix (ECM) production are significantly decreased in arterial walls and VSMCs of MFG-E8 knockout mice. Taken together, these findings indicate that MFG-E8 is the key modulator of the TGF-1/SMAD/collagen signaling cascade in VSMCs, likely contributing to arterial stiffening with aging. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for prevention and treatment of the quintessential age-associated diseases, i.e., arthrosclerosis and diseases involved in inflammation and smooth muscle cell invasion, proliferation, and extracellular remodeling.